Project Summary/Abstract HLA-F is a nonclassical class I MHC (Ib) molecule that has been found expressed on a variety of cancers, shown to play a role in HIV infection and the neurological autoimmune disease ALS, and is expressed throughout pregnancy. Despite the potential importance of this protein in these conditions, little is known about this molecule in terms of its function or even in which state it is expressed. We have recently shown that, in addition to being expressed as a heavy chain only state, or open conformer (HLA-FOC), HLA-F can also be expressed as a bona fide peptide presenting molecule, associated with the ?2m subunit (pHLA-F). Peptides are presented in an unconventional way, with the N-terminus not anchored within the groove and the potential for post-translational modifications featuring in peptide anchoring. Despite these advances, there remains much unknown about the role for presented peptide in HLA-F function and how HLA-F engages its various receptors in each of these states. Thus, the aims of this proposal focus on addressing these questions and are: Aim 1: Develop and validate nanobodies towards peptide-loaded (pHLA-F) and open conformer (HLA- FOC) states of HLA-F and determine their expression profile in cancer cell lines. As HLA-F has been described to function in both the peptide-bound and free states, we will develop and validate novel nanobodies that specifically bind to these different forms of HLA-F. We will validate them through the use of HLA-F over expressing and knock out cell lines by flow cytometry, immunoprecipitation, and western blot. Finally, validated nanobody-binders will be used to determine the dominant form of HLA-F (HLA-FOC, pHLA-F, or both) expressed in and on the surface of 1) standard cell lines and 2) from gynecologic, neuroblastoma, and hepatocellular carcinoma cancer cell lines. Aim 2: Determine the peptide-antigen repertoire of HLA-F from selected cancer cell lines and determine their effect on NK-cell receptor engagement. As we have previously published, we have the ability to produce recombinant pHLA-F from HEK293T cells using an engineered baculovirus. We also showed that we can use this purified protein to determine the peptide repertoire of HLA-F from this cell line using MS/MS. Using this approach, we will produce pHLA-F from the cancer cell lines mentioned in SA1 and determine the peptide repertoire from these cancer specific lineages. In addition, we will pull out endogenous protein from these lines and confirm our results or act as an alternative approach should recombinant protein prove difficult to produce. Finally, we will determine the role of these peptide-antigens in NK-cell receptor engagement using in vitro binding experiments.